Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.